The development of transformative therapeutics, including the possibility of a cure though gene therapy, continues to be the major research and development activity in the commercial hemophilia space. For example, there are several company-sponsored ongoing clinical trials of recombinant adeno-associated viral (rAAV) encoding coagulation factor IX (FIX) for the treatment of hemophilia A. Furthermore as a result of these trials, there now exists extensive safety and initial efficacy data for multiple rAAV-FIX products. However, progress in the translation of this approach to hemophilia A has been hampered by significant hurdles including the size, complexity, immunogenicity, instability and biosynthetic inefficiency of coagulation factor VIII (FVIII) in comparison to FIX. Now through the identification of 1) liver-specific transcriptional regulatory modules (LTRMs) that are smaller tha existing enhancer-promoters combinations and 2) a liver-enhanced codon- optimization algorithm used to engineer the coagulation factor VIII (FVIII) transgene, we have been able to design rAAV vectors encoding FVIII with genome sizes under 5,000 nucleotides that have unprecedented potency. The proposed milestones of the current project are 1) to characterize and select a lead candidate LTRM optimized for FVIII mRNA and transgene product output, rAAV vector manufacture yield and rAAV product consistency, and 2) to identify a lead candidate AAV-FVIII transgene. These proof of concept studies will be performed using the preclinical murine model of hemophilia A. The data obtained should facilitate the selection and preclinical validation of a lead candidate liver-directed AAV-FVIII expression cassette combining these novel technologies exclusively available to Expression Therapeutics (ET). Therefore, ET is in an opportunistic position to capitalize on the ongoing rAAV-FIX/hemophilia B clinical findings and incorporate preclinical R&D and clinical protocol development strategies into this high priority rAAV-FVIII project. This existing clinical gene therapy knowledge combined with our hemophilia A experience and innovative technologies should enable ET to overcome the major hurdles facing commercialization of liver-directed rAAV-FVIII therapy for hemophilia A.